Flexibility of Eukaryotic Okazaki Fragment Maturation through Regulated Strand Displacement Synthesis
Flexibility of Eukaryotic Okazaki Fragment Maturation through Regulated Strand Displacement Synthesis
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DOI：
10.1074/jbc.m806668200
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发表时间：
2008-12-05
影响因子：
4.8
通讯作者：
Burgers, Peter M.
中科院分区：
文献类型：
--
作者：
Stith, Carrie M.;Sterling, Joan;Burgers, Peter M.
为了在真核细胞中产生连续的滞后链，Okazaki片段的成熟需要DNA聚合酶Delta(Pol Delta)与FEN1(RAD27)内切酶切割的5‘-翻盖的精确协调。通常，Pol Delta的3‘-核酸外切酶活性可以防止过度的链移位。这种核心成熟机制可以由处理长襟翼的DNA2核酸酶/解旋酶辅助。我们的遗传学研究表明，POL32(POL Delta第三亚单位)或PIF1解旋酶基因的缺失可以抑制产生长瓣的rad27 Delta pol3-D520V突变体(FEN1RAD27和Pol Delta的3‘-核酸外切酶缺陷)的致死性或生长缺陷，以及DNA2 Delta突变体在切割长瓣方面存在缺陷。相反，pol32 Delta或pif1 Delta导致了rad27 Delta Exo1 Delta双重突变体的致死，这表明在缺乏FEN1(RAD27)和Exo1的短襟翼处理活动的情况下，Pol32和Pif1需要产生可被DNA2处理的较长襟翼。遗传分析表明冈崎成熟机械具有显著的灵活性，与我们的生化分析一致。在体外，POL Delta产生的短襟翼不受Pol32的存在的影响；然而，只有当Pol32存在时，较长的襟翼才会积累。在链置换合成过程中，FEN1(RAD27)的存在减少了位移，有利于瓣片切割，从而表明了从Pol Delta到FEN1(RAD27)的主动移交机制。最后，RNA-DNA杂交体比DNA杂交体更容易被Pol Delta取代，从而有利于启动子RNA在Okazaki成熟过程中的降解。
Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase delta (Pol delta) with 5'-flap cutting by FEN1(RAD27) endonuclease. Excessive strand displacement is normally prevented by the 3' -exonuclease activity of Pol delta. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol delta) or PIF1 helicase genes can suppress lethality or growth defects of rad27 Delta pol3-D520V mutants (defective for FEN1RAD27 and the 3' -exonuclease of Pol delta) that produce long flaps and of dna2 Delta mutants that are defective in cutting long flaps. On the contrary, pol32 Delta or pif1 Delta caused lethality of rad27 Delta exo1 Delta double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1(RAD27) and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol delta is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1(RAD27) during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol delta to FEN1(RAD27). Finally, RNA-DNA hybrids are more readily displaced by Pol delta than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.